TCRs mediate the recognition of specific pMHC by T cells and, as such, are essential to the functioning of the cellular arm of the immune system. The native TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have quite distinct anatomical locations and probably functions. The MHC class I and class II ligands are also immunoglobulin superfamily proteins but are specialised for antigen presentation, with a highly polymorphic peptide binding site which enables them to present a diverse array of short peptide fragments at the antigen presenting cell (“APC”) surface.
Two further classes of proteins are known to be capable of functioning as TCR ligands, (1) CD1 antigens are MHC class I-related molecules whose genes are located on a different chromosome from the classical MHC class I and class II antigens. CD1 molecules are capable of presenting peptide and non-peptide (eg lipid, glycolipid) moieties to T cells in a manner analogous to conventional class I and class II-MHC-peptide complexes. See, for example (Barclay et al, (1997) The Leucocyte Antigen Factsbook 2nd Edition, Academic Press) and (Bauer (1997) Eur J Immunol 27 (6) 1366-1373)). Bacterial superantigens are soluble toxins which are capable of binding both class II MHC molecules and a subset of TCRs. (Fraser (1989) Nature 339 221-223). Many superantigens exhibit specificity for one or two Vbeta segments, whereas others exhibit more promiscuous binding. In any event, superantigens are capable of eliciting an enhanced immune response by virtue of their ability to stimulate subsets of T cells in a polyclonal fashion.
The extracellular portion of native heterodimeric αβ and γδ TCRs consist of two polypeptides each of which has a membrane-proximal constant domain, and a membrane-distal variable domain. Each of the constant and variable domains includes an intra-chain disulfide bond. The variable domains contain the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies. CDR3 of αβ TCRs predominantly interact with the peptide presented by MHC, and CDRs 1 and 2 of αβ TCRs predominantly interact with the peptide and the MHC. The diversity of TCR variable domain sequences is generated via somatic rearrangement of linked variable (V), diversity (D), joining (J), and constant genes
Functional α and γ chain TCR polypeptides are formed by rearranged V-J-C regions, whereas β and δ chains consist of V-D-J-C regions. The extracellular constant domain has a membrane proximal region and an immunoglobulin region. There are single a and δ chain constant domains, known as TRAC and TRDC respectively and the β chain constant domain is composed of one of two different β constant domains, known as TRBC1 and TRBC2 (IMGT nomenclature). There are four amino acid changes between these β constant domains, three of which are within the domains used to produce the single-chain TCRs displayed on phage particles of the present invention. These changes are all within exon 1 of TRBC1 and TRBC2: N4K5->K4N5 and F37->Y (IMGT numbering, differences TRBC1->TRBC2), the final amino acid change between the two TCR β chain constant regions being in exon 3 of TRBC1 and TRBC2: V1->E. The constant γ domain is composed of one of either TRGC1, TRGC2 (2×) or TRGC2 (3×). The two TRGC2 constant domains differ only in the number of copies of the amino acids encoded by exon 2 of this gene that are present.
The extent of each of the TCR extracellular domains is somewhat variable. However, a person skilled in the art can readily determine the position of the domain boundaries using a reference such as The T Cell Receptor Facts Book, Lefranc & Lefranc, Publ. Academic Press 2001.
TCRs can be prepared by recombinant means. A number of constructs have been devised to date for the production of recombinant TCRs. These constructs fall into two broad classes, single-chain TCRs (“scTCRs”) and dimeric TCRs (“dTCRs”).
Display Methods
Particle display methods have primarily been used to identify proteins with desirable properties such as enhanced expression yields, binding and/or stability characteristics. These methods involve creating a diverse pool or ‘library’ of proteins or polypeptides expressed on the surface of nucleoprotein particles. These particles have two key features, firstly each particle presents a single variant protein or polypeptide, and secondly the genetic material encoding the expressed protein or polypeptide is associated with that of the particle. This library is then subjected to one or more rounds of selection. For example, this may consist of contacting a ligand with a particle-display library of mutated receptors and identifying which mutated receptors bind the ligand with the highest affinity. Once the selection process has been completed the receptor or receptors with the desired properties can be isolated, and their genetic material can be amplified in order to allow the receptors to be sequenced.
Particularly preferred is the phage display technique which is based on the ability of bacteriophage particles to express a heterologous peptide or polypeptide fused to their surface proteins. (Smith (1985) Science 217 1315-1317). The procedure is quite general, and well understood in the art for the display of polypeptide monomers. However, in the case of polypeptides that in their native form associate as dimers, only the phage display of antibodies appears to have been thoroughly investigated.
For monomeric polypeptide display there are two main procedures:
Firstly (Method A) by inserting into a vector (phagemid) DNA encoding the heterologous peptide or polypeptide fused to the DNA encoding a bacteriophage coat protein. The expression of phage particles displaying the heterologous peptide or polypeptide is then carried out by transfecting bacterial cells with the phagemid, and then infecting the transformed cells with a ‘helper phage’. The helper phage acts as a source of the phage proteins not encoded by the phagemid required to produce a functional phage particle.
Secondly (Method B), by inserting DNA encoding the heterologous peptide or polypeptide into a complete phage genome fused to the DNA encoding a bacteriophage coat protein. The expression of phage particles displaying the heterologous peptide or polypeptide is then carried out by infecting bacterial cells with the phage genome. This method has the advantage of the first method of being a ‘single-step’ process. However, the size of the heterologous DNA sequence that can be successfully packaged into the resulting phage particles is reduced. M13, T7 and Lambda are examples of suitable phages for this method.
A variation on (Method B) the involves adding a DNA sequence encoding a nucleotide binding domain to the DNA in the phage genome encoding the heterologous peptide be displayed, and further adding the corresponding nucleotide binding site to the phage genome. This causes the heterologous peptide to become directly attached to the phage genome. This peptide/genome complex is then packaged into a phage particle which displays the heterologous peptide. This method is described in WO 99/11785.
The phage particles can then be recovered and used to study the binding characteristics of the heterologous peptide or polypeptide. Once isolated, phagemid or phage DNA can be recovered from the peptide- or polypeptide-displaying phage particle, and this DNA can be replicated via PCR. The PCR product can be used to sequence the heterologous peptide or polypeptide displayed by a given phage particle.
The phage display of single-chain antibodies and fragments thereof, has become a routine means of studying the binding characteristics of these polypeptides. There are numerous books available that review phage display techniques and the biology of the bacteriophage. (See, for example, Phage Display—A Laboratory Manual, Barbas et al., (2001) Cold Spring Harbour Laboratory Press).
A third phage display method (Method C) relies on the fact that heterologous polypeptides having a cysteine residue at a desired location can be expressed in a soluble form by a phagemid or phage genome, and caused to associate with a modified phage surface protein also having a cysteine residue at a surface exposed position, via the formation of a disulphide linkage between the two cysteines. WO 01/05950 details the use of this alternative linkage method for the expression of single-chain antibody-derived peptides.
High Affinity TCRs
T cells mature in the thymus where they undergo at least two selection mechanisms, generally referred to as positive and negative selection. The structures of most, or all, TCRs are believed to share certain general architectural features (Chothia, et al, Embo J (1988) 7: 3745-55) that provide a framework suitable for MHC/peptide binding by the variable complementarity determining regions (CDRs). Thus, most TCRs may have intrinsic affinity for MHC/peptide complexes (Chothia, et al, Embo J (1988) 7: 3745-55). In the thymus, only TCRs with a certain minimal level of affinity for one of the MHC molecules to which they are presented (the “self” MHC molecules) will be positively selected. T cells with high affinity for one of the self MHC molecules will be negatively selected (Amsen & Kruisbeek. (1998). Immunol Rev 165: 209-29. Sebzda, et al (1999). Annu Rev Immunol 17: 829-74).
TCRs in the cellular immunity can be considered to be analogous to antibodies in the humoral immunity. Antibodies have been successfully used, either as therapeutic agents in their own right (e.g. Herceptin) or as targeting agents (e.g. mylotarg), and interest in this area continues to grow. Similar strategies could be devised using T cell receptors. Thus, soluble TCRs are useful, not only for the purpose of investigating specific TCR-pMHC interactions, but also as a diagnostic tool to detect infection, or to detect autoimmune disease markers, or to detect the efficacy of T cell vaccines. Soluble TCRs also have applications in staining, for example to stain cells for the presence of a particular viral antigen presented in the context of the MHC. Similarly, soluble TCRs can be used to deliver a therapeutic agent, for example a cytotoxic compound or an immunostimulating compound, to cells presenting a particular antigen.
However, two factors have hindered the exploitation of TCRs in this way. Firstly, a generally applicable method for the production of soluble (i.e. non-membrane bound) T cell receptors has not been available until recently. Secondly, the affinity of the T cell receptor for its specific pMHC ligand is much lower (KD in the μM range) than for antibodies (KD in the nM range). This lower affinity of the TCR is thought to be a result of negative selection during development, and it is therefore probably not possible to find TCRs with high affinity for self-MHC-peptide complexes (Salzmann & Bachmann, Molecular Immunology, 1998, 35:65-71